Triazine compounds and methods of use therefor

ABSTRACT

The present invention relates to compositions and methods for inhibiting nonenzymatic cross-linking (protein aging). Accordingly, a composition is disclosed which comprises a substituted 1,2,4-triazine of the formula (I) ##STR1## and the biologically and pharmaceutically acceptable salts thereof; capable of inhibiting the formation of advanced glycosylation endproducts of target proteins. The method comprises contacting the target protein with the composition. Both industrial and therapeutic applications for the invention are envisioned, as food spoilage and animal protein aging can be treated.

Continuation of Ser. No. 08/825,114, filed Mar. 27, 1997, now U.S. Pat.No. 5,932,578, which claims priority from U.S. provisional Applications60/014,387, filed Mar. 28, 1996 and 60/016,822, filed May 3, 1996.

BACKGROUND OF THE INVENTION

The present invention relates generally to the aging of proteinsresulting from their reaction with glucose and other reducing sugars,and more particularly to the inhibition of the reaction ofnonenzymatically glycosylated proteins and the often resultant formationof advanced glycosylation (glycation) endproducts and cross-links.

The reaction between glucose and proteins has been known for some time.Its earliest manifestation was in the appearance of brown pigmentsduring the cooking of food, which was identified by Maillard in 1912,who observed that glucose or other reducing sugars react with aminoacids to form adducts that undergo a series of dehydrations andrearrangements to form stable brown pigments. Further studies havesuggested that stored and heat treated foods undergo nonenzymaticbrowning as a result of the reaction between glucose and the polypeptidechain, and that the proteins are resultingly cross-linked andcorrespondingly exhibit decreased bioavailability.

This reaction between reducing sugars and food proteins was found tohave its parallel in vivo. Thus, the nonenzymatic reaction betweenglucose and the free amino groups on proteins to form a stable,1-deoxyketosyl adduct, known as the Amadori product, has been shown tooccur with hemoglobin, wherein a rearrangement of the amino terminal ofthe beta-chain of hemoglobin by reaction with glucose, forms the adductknown as hemoglobin A1c. The reaction has also been found to occur witha variety of other body proteins, such as lens crystallins, collagen andnerve proteins. See Bucala et al., "Advanced Glycosylation; Chemistry,Biology, and Implications for Diabetes and Aging" in Advances inPharmacology, Vol. 23, pp. 1-34, Academic Press (1992).

Moreover, brown pigments with spectral and fluorescent propertiessimilar to those of late-stage Maillard products have also been observedin vivo in association with several long-lived proteins, such as lensproteins and collagen from aged individuals. An age-related linearincrease in pigment was observed in human dura collagen between the agesof 20 to 90 years. Interestingly, the aging of collagen can be mimickedin vitro by the cross-linking induced by glucose; and the capture ofother proteins and the formation of adducts by collagen, also noted, istheorized to occur by a cross-linking reaction, and is believed toaccount for the observed accumulation of albumin and antibodies inkidney basement membrane.

In U.S. Pat. No. 4,758,583, a method and associated agents weredisclosed that served to inhibit the formation of advanced glycosylationendproducts by reacting with an early glycosylation product that resultsfrom the original reaction between the target protein and glucose.Accordingly, inhibition was postulated to take place as the reactionbetween the inhibitor and the early glycosylation product appeared tointerrupt the subsequent reaction of the glycosylated protein withadditional protein material to form the cross-linked late-stage product.One of the agents identified as an inhibitor was aminoguanidine, and theresults of further testing have bone out its efficacy in this regard.

While the success that has been achieved with aminoguanidine and similarcompounds is promising, a need continues to exist to identify anddevelop additional inhibitors that broaden the availability and perhapsthe scope of this potential activity and its diagnostic and therapeuticutility.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method and compositions aredisclosed for the inhibition of the advanced glycosylation of proteins(protein aging). In particular, the compositions comprise agents forinhibiting nonenzymatic cross-linking (protein aging) due to theformation of advanced glycosylation (glycation) endproducts. The agentsmay be selected from those materials capable of reacting with an earlyglycosylation product from the reaction of glucose with proteins andpreventing further reactions. Cross-linking caused by other reactivesugars present in vivo or in foodstuffs, including ribose, galactose andfructose would also be prevented by the methods and compositions of thepresent invention.

The agents comprise substituted triazine compounds having the followingstructural formula: ##STR2## wherein R is hydrogen or an amino group; R'is a cycloalkyl group;

an aralkyl group wherein the alkyl portion contains from 1 to 6 carbonatoms, and the aryl portion is optionally substituted by one to threehydroxy, lower alkoxy or nitro groups;

a heteroaryl group containing from 1 to 3 heteroatoms selected from thegroup consisting of sulfur, oxygen and nitrogen;

a group of the formula ##STR3## wherein R" is hydroxy, a lower alkoxygroup, an aryl group optionally substituted by a lower alkoxy group, ora group of the formula --NR"'NH₂ wherein R"' is hydrogen or a methylgroup; or

a lower alkyl or alkylene group, optionally substituted by hydroxy or agroup of the formula ##STR4## wherein R" is hydroxy, a lower alkoxygroup, an aryl group optionally substituted by a lower alkoxy group, ora group of the formula --NR"'NH₂ wherein R"' is hydrogen or a methylgroup;

and their biologically or pharmaceutically acceptable acid additionsalts; and mixtures thereof, and a carrier therefor.

The compounds, and their compositions, utilized in this invention appearto react with an early glycosylation product thereby preventing the samefrom later forming the advanced glycosylation end products which lead toprotein cross-links, and thereby, to protein aging.

The present invention also relates to a method for inhibiting proteinaging by contacting the initially glycosylated protein at the stage ofthe early glycosylation product with a quantity of one or more of theagents of the present invention, or a composition containing the same.In the instance where the present method has industrial application, oneor more of the agents may be applied to the proteins in question, eitherby introduction into a mixture of the same in the instance of a proteinextract, or by application or introduction into foodstuffs containingthe protein or proteins, all to prevent premature aging and spoilage ofthe particular foodstuffs.

The ability to inhibit the formation of advanced glycosylationendproducts carries with it significant implications in all applicationswhere protein aging is a serious detriment. Thus, in the area of foodtechnology, the retardation of food spoilage would confer an obviouseconomic and social benefit by making certain foods of marginalstability less perishable and therefore more available for consumers.Spoilage would be reduced as would the expense of inspection, removal,and replacement, and the extended availability of the foods could aid instabilizing their price in the marketplace. Similarly, in otherindustrial applications where the perishability of proteins is aproblem, the admixture of the agents of the present invention incompositions containing such proteins would facilitate the extendeduseful life of the same. Presently used food preservatives anddiscoloration preventatives such as sulfur dioxide, known to causetoxicity including allergy and asthma in animals, can be replaced withcompounds such as those described herein.

The present method has particular therapeutic application as theMaillard process acutely affects several of the significant proteinmasses in the body, among them collagen, elastin, lens proteins, and thekidney glomerular basement membranes. These proteins deteriorate bothwith age (hence the application of the term "protein aging") and as aconsequence of diabetes. Accordingly, the ability to either retard orsubstantially inhibit the formation of advanced glycosylationendproducts carries the promise of treatment for diabetes and, ofcourse, improving the quality and, perhaps, duration of animal life.

The present agents are also useful in the area of personal appearanceand hygiene, as they prevent the staining of teeth by cationicanti-microbial agents with anti-plaque properties, such aschlorhexidine.

Accordingly, it is a principal object of the present invention toprovide a method for inhibiting the extensive cross-linking of proteinsthat occurs as an ultimate consequence of the reaction of the proteinswith glucose and other reactive sugars, by correspondingly inhibitingthe formation of advanced glycosylation endproducts.

It is a further object of the present invention to provide a method asaforesaid which is characterized by a reaction with an initiallyglycosylated protein identified as an early glycosylation product.

It is a further object of the present invention to provide a method asaforesaid which prevents the rearrangement and cross-linking of the saidearly glycosylation products to form the said advanced glycosylationendproducts.

It is a yet further object of the present invention to provide agentscapable of participating in the reaction with the said earlyglycosylation products in the method as aforesaid.

It is a still further object of the present invention to providetherapeutic methods of treating the adverse consequences of proteinaging by resort to the aforesaid method and agents.

It is a still further object of the present invention to provide amethod of inhibiting the discoloration of teeth by resort to theaforesaid method and agents.

It is a still further object of the present invention to providecompositions including pharmaceutical compositions, all incorporatingthe agents of the present invention.

It is still further object of the present invention to provide novelcompounds, as well as processes for their preparation, for use in themethods and compositions of the present invention.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the present invention, agents, compositions includingpharmaceutical compositions containing said agents and associatedmethods have been developed which are believed to inhibit the formationof advanced glycosylation endproducts in a number of target proteinsexisting in both animals and plant material. In particular, theinvention relates to a composition which may contain one or more agentscomprising substituted triazine compounds having the structural formula:##STR5## wherein R is hydrogen or an amino group; R' is a cycloalkylgroup;

an aralkyl group wherein the alkyl portion contains from 1 to 6 carbonatoms, and the aryl portion is optionally substituted by one to threehydroxy, lower alkoxy or nitro groups;

a heteroaryl group containing from 1 to 3 heteroatoms selected from thegroup consisting of sulfur, oxygen and nitrogen;

a group of the formula ##STR6## wherein R" is hydroxy, a lower alkoxygroup, an aryl group optionally substituted by a lower alkoxy group, ora group of the formula --NR"'NH₂ wherein R"' is hydrogen or a methylgroup; or

a lower alkyl or alkylene group, optionally substituted by hydroxy or agroup of the formula ##STR7## wherein R" is hydroxy, a lower alkoxygroup, an aryl group optionally substituted by a lower alkoxy group, ora group of the formula --NR"'NH₂ wherein R"' is hydrogen or a methylgroup;

and their biologically or pharmaceutically acceptable acid additionsalts; and mixtures thereof, and a carrier therefor.

The lower alkyl groups referred to above preferably contain 17 carbonatoms and include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyland the corresponding branched-chain isomers thereof. The lower alkylenegroups likewise contain from 2 to 6 carbon atoms, and are exemplified byethylene, propylene, butylene, pentylene, hexylene, and thecorresponding branched chain isomers thereof. These groups areoptionally substituted by one or more hydroxy groups, or a group of theformula ##STR8## wherein R" is hydroxy, a lower alkoxy group, an arylgroup optionally substituted by a lower alkoxy group, or a group of theformula --NR"' NH₂ wherein R"' is hydrogen or a methyl group.

These substituted carbonyl compounds are thus carboxy, alkoxycarbonyl,arylcarbonyl or hydrazinylcarbonyl compounds wherein the alkyl portioncontains from 1 to 6 carbon atoms as described hereinabove. Similarly,the aryl portion is as defined hereinbelow.

The aryl groups encompassed by the above formula are those containing6-10 carbon atoms, such as naphthyl, phenyl and lower alkylsubstituted-phenyl, e.g., tolyl and xylyl, and are optionallysubstituted by 1-3 hydroxy, lower alkoxy or nitro groups. Preferred arylgroups are phenyl and methoxyphenyl groups.

Where the possibility exists for substitution of a phenyl or aryl ring,the position of the substituents may be ortho, meta, or para to thepoint of attachment of the phenyl or aryl ring to the nitrogen of thehydrazine group.

The cycloalkyl groups contain from 1 to 7 carbon atoms, and are typifiedby cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl,optionally substituted by one or more lower alkyl groups.

The heteroaryl groups of the compounds of formula I typically containfrom 5 to 6 ring atoms, of which 1 to 3 are selected from the groupconsisting of oxygen, nitrogen and sulfur. Representative of such groupsare 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-thienyl, 3-thienyl, 2-furanyl,3-furanyl, 2-pyrrolyl, and 3-pyrrolyl, optionally substituted by one ormore lower alkyl groups.

The lower alkoxy groups contain 1-6, and preferably 1-3, carbon atomsand are illustrated by methoxy, ethoxy, n-propoxy, isopropoxy and thelike.

For the purposes of this invention equivalent to the compounds offormula (I) are the biologically and pharmaceutically acceptable acidaddition salts thereof. Such acid addition salts may be derived from avariety of organic and inorganic acids such as sulfuric, phosphoric,hydrochloric, hydrobromic, sulfamic, citric, lactic, maleic, succinic,tartaric, cinnamic, acetic, benzoic, gluconic, ascorbic, methanesulfonicand related acids.

Of the compounds encompassed by Formula I, certain substituents arepreferred. For instance, the compounds wherein R' is a lower alkylgroup, and particularly those wherein R' is a methyl group, arepreferred.

Representative compounds of the present invention are:

3,4-diamino-6-methyl-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-(4-hydroxybenzyl)-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-ethyl-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-benzyl-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-(2-thienyl)-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-(3,4-dimethoxystyryl)-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-(2'-nitrobenzyl)-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-(2'-carboxyethyl)-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-isopropyl-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-(2-methypropyl)-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-propyl-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-butyl-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-(1-methylpropyl)-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-cyclopropyl-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-phenethyl-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-carboxyethyl-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-(2'-ethoxycarbonylethyl)-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-(1'-ethoxycarbonylethyl)-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-(1'-ethoxycarbonylheptyl)-1,2,4-triazine-5(4H)-one;

3,4-diamino-6-(3'-methoxyphenylcarbonylmethylene)-1,2,4-triazine-5(4H)-one;

6-(3,4-diamino-5(4H)-one-1,2,4-triazinyl)hydrazide;

3-[6'-(3',4'-diamino-5'(4H)-one-1',2',4'-triazinyl)]-propionichydrazide;

2-methyl-6'-(3',4'-diamino-5(4H)-one-1,2,4-triazinyl)hydrazide;

3,4-diamino-6-(1',1'-dimethyl-2'-hydroxyethyl)-1,2,4triazine-5(4H)-one;

4-amino-3-hydrazino-6-methyl-1,2,4-triazine-5(4H)-one;

4-amino-3-hydrazino-6-(4-hydroxybenzyl)-1,2,4-triazine-5(4H)-one;

4-amino-3-hydrazino-1,2,4-triazine-5(4H)-one; and their biologically andpharmaceutically acceptable salts.

The above compounds are capable of inhibiting the formation of advancedglycosylation endproducts on target proteins. The cross-linking of theprotein to form the advanced glycosylation endproduct contributes to theentrapment of other proteins and results in the development in vivo ofconditions such as reduced elasticity and wrinkling of the skin, certainkidney diseases, atherosclerosis, osteoarthritis and the like.Similarly, plant material that undergoes nonenzymatic browningdeteriorates and, in the case of foodstuffs, become spoiled or toughenedand, consequently, inedible. Thus, the compounds employed in accordancewith this invention inhibit this late-stage Maillard effect andintervene in the deleterious changes described above.

The rationale of the present invention is to use agents which block thepost-glycosylation step, i.e., the formation of fluorescentchroinophores, the presence of which chromophores is associated with,and leads to adverse sequelae of diabetes and aging. An ideal agentwould prevent the formation of the chromophore and its associatecross-links of proteins to proteins and trapping of proteins on theother proteins, such as occurs in arteries and in the kidney.

The chemical nature of the early glycosylation products with which thecompounds of the present invention are believed to react may vary, andaccordingly the term "early glycosylation product(s)" as used herein isintended to include any and all such variations within its scope. Forexample, early glycosylation products with carbonyl moieties that areinvolved in the formation of advanced glycosylation endproducts, andthat may be blocked by reaction with the compounds of the presentinvention, have been postulated. In one embodiment, it is envisionedthat the early glycosylation product may comprise the reactive carbonylmoieties of Amadori products or their further condensation, dehydrationand/or rearrangement products, which may condense to form advancedglycosylation endproducts. In another scenario, reactive carbonylcompounds, containing one or more carbonyl moieties (such asglycolaldehyde, glyceraldehyde or 3-deoxyglucosone) may form from thecleavage of Amadori or other early glycosylation endproducts, and bysubsequent reactions with an amine or Amadori product, may form carbonylcontaining advanced glycosylation products such asalkylformyl-glycosylpyrroles.

The compositions useful in the present invention comprise or containagents capable of reacting with the active carbonyl intermediate of anearly glycosylation product. Suitable agents are the compounds ofFormula I of the present invention. The present invention likewiserelates to methods for inhibiting the formation of advancedglycosylation endproducts, which comprise contacting the target proteinswith a composition of the present invention. In the instance where thetarget proteins are contained in foodstuffs, whether of plant or animalorigin, these foodstuffs could have applied to them by variousconventional means a composition containing the present agents.

In the food industry, sulfites were found years ago to inhibit theMaillard reaction and are commonly used in processed and stored foods.Recently, however, sulfites in food have been implicated in severe andeven fatal reactions in asthmatics. As a consequence, the sulfitetreatment of fresh fruits and vegetables has been banned. The mechanismfor the allergic reaction is not known. Accordingly, the presentcompositions and agents offer a nontoxic alternative to sulfites in thetreatment of foods in this manner.

As is apparent from a discussion of the environment of the presentinvention, the present methods and compositions hold the promise forarresting the aging of key proteins both in animals and plants, andconcomitantly, conferring both economic and medical benefits as a resultthereof. In the instance of foodstuffs, the administration of thepresent composition holds the promise for retarding flood spoilagethereby making foodstuffs of increased shelf life and greateravailability to consumers. Replacement of currently-used preservatives,such as sulfur dioxide known to cause allergies and asthma in humans,with non-toxic, biocompatible compounds is a further advantage of thepresent invention.

The therapeutic implications of the present invention relate to thearrest of the aging process which has, as indicated earlier, beenidentified in the aging of key proteins by advanced glycosylation andcross-linking. Thus, body proteins, and particularly structural bodyproteins, such as collagen, elastin, lens proteins, nerve proteins,kidney glomerular basement membranes and other extravascular matrixcomponents would all benefit in their longevity and operation from thepractice of the present invention. The present invention thus reducesthe incidence of pathologies involving the entrapment of proteins bycross-linked target proteins, such as retinopathy, cataracts, diabetickidney disease, glomerulosclerosis, peripheral vascular disease,arteriosclerosis obliterans, peripheral neuropathy, stroke,hypertension, atherosclerosis, osteoarthritis, periarticular rigidity,loss of elasticity and wrinkling of skin, stiffening of joints,glomerulonephritis, etc. Likewise, all of these conditions are inevidence in patients afflicted with diabetes mellitus. Thus, the presenttherapeutic method is relevant to treatment of the noted conditions inpatients either of advanced age or those suffering from one of thementioned pathologies.

Protein cross-linking through advanced glycosylation product formationcan decrease solubility of structural proteins such as collagen invessel walls and can also trap serum proteins, such as lipoproteins tothe collagen. Also, this may result in increased permeability of theendothelium and consequently covalent trapping of extravasated plasmaproteins in subendothelial matrix, and reduction in susceptibility ofboth plasma and matrix proteins to physiologic degradation by enzymes.For these reasons, the progressive occlusion of diabetic vessels inducedby chronic hyperglycemia has been hypothesized to result from excessiveformation of glucose-derived cross-links. Such diabetic microvascularchanges and microvascular occlusion can be effectively prevented bychemical inhibition of advanced glycosylation product formationutilizing a composition and the methods of the present invention.

Studies indicate that the development of chronic diabetic damage intarget organs is primarily linked to hyperglycemia so that tightmetabolic control would delay or even prevent end-organ damage. SeeNicholls et al., Lab. Invet., 60, No. 4, p. 486 (1989), which discussesthe effects of islet isografting and aminoguanidine in murine diabeticnephropathy. These studies further evidence that aminoguanidinediminishes aortic wall protein cross-linking in diabetic rats andconfirm earlier studies by Brownlee et al., Science, 232, pp. 1629-1632(1986) to this additional target organ of complication of diabetes.Also, an additional study showed the reduction of immunoglobulintrapping in the kidney by aminoguanidine (Brownlee et al., Diabetes, 35,Suppl. 1, p. 42A (1986)).

Further evidence in the streptozotocin-diabetic rat model thataminoguanidine administration intervenes in the development of diabeticnephropathy was presented by Brownlee et al., 1988, supra, with regardto morphologic changes in the kidney which are hallmarks of diabeticrenal disease. These investigators reported that the increasedglomerular basement membrane thickness, a major structural abnormalitycharacteristic of diabetic renal disease, was prevented withaminoguanidine.

Taken together, these data strongly suggest that inhibition of theformation of advanced glycosylation endproducts (AGEs), by the teachingof the present invention, may prevent late, as well as early, structurallesions due to diabetes, as well as changes during aging caused by theformation of AGEs.

Diabetes-induced changes in the deformability of red blood cells,leading to more rigid cell membranes, is another manifestation ofcross-linking and aminoguanidine has been shown to prevent it in vivo.In such studies, New Zealand White rabbits, with induced, long-termdiabetes are used to study the effects of a test compound on red bloodcell (RBC) deformability (df). The test compound is administered at arate of 100 mg/kg by oral gavage to diabetic rabbits.

A further consequence of diabetes is the hyperglycemia-induced matrixbone differentiation resulting in decreased bone formation usuallyassociated with chronic diabetes. In animal models, diabetes reducesmatrix-induced bone differentiation by 70%.

In the instance where the compositions of the present invention areutilized for in vivo or therapeutic purposes, it may be noted that thecompounds or agents used therein are biocompatible. Pharmaceuticalcompositions may be prepared with a therapeutically effective quantityof the agents or compounds of the present invention and may include apharmaceutically acceptable carrier, selected from known materialsutilized for this purpose. Such compositions may be prepared in avariety of forms, depending on the method of administration. Also,various pharmaceutically acceptable addition salts of the compounds ofFormula I may be utilized.

A liquid form would be utilized in the instance where administration isby intravenous, intramuscular or intraperitoneal injection. Whenappropriate, solid dosage forms such as tablets, capsules, or liquiddosage formulations such as solutions and suspensions, etc., may beprepared for oral administration. For topical or dermal application tothe skin or eye, a solution, a lotion or ointment may be formulated withthe agent in a suitable vehicle such as water, ethanol, propyleneglycol, perhaps including a carrier to aid in penetration into the skinor eye. For example, a topical preparation could include up to about 10%of the compound of Formula I. Other suitable forms for administration toother body tissues are also contemplated.

In the instance where the present method has therapeutic application,the animal host intended for treatment may have administered to it aquantity of one or more of the agents, in a suitable pharmaceuticalform. Administration many be accomplished by known techniques, such asoral, topical and parenteral techniques such as intradermal,subcutaneous, intravenous or intraperitoneal injection, as well as byother conventional means. Administration of the agents may take placeover an extended period of time at a dosage level of, for example, up toabout 30 mg/kg.

As noted earlier, the invention also extends to a method of inhibitingthe discoloration of teeth resulting from nonenzymatic browning in theoral cavity which comprises administration to a subject in need of suchtherapy an amount effective to inhibit the formation of advancedglycosylation endproducts of a composition comprising an agent ofstructural Formula I.

The nonenzymatic browning reaction which occurs in the oral cavityresults in the discoloration of teeth. Presently used anti-plaque agentsaccelerate this nonenzymatic browning reaction and further the stainingof the teeth. Recently, a class of cationic anti-microbial agents withremarkable anti-plaque properties have been formulated in oral rinsesfor regular use to kill bacteria in the mouth. These agents, thecationic antiseptics, include such agents as alexidine, cetyl pyridiniumchloride, chlorhexidine gluconate, hexetidine, and benzalkoniumchloride.

Tooth staining by chlorhexidine and other anti-plaque agents apparentlyresults from the enhancement of the Maillard reaction. Nordbo, J. Dent.Res., 58, p. 1429 (1979) reported that chlorhexidine and benzalkoniumchloride catalyze browning reactions in vitro. Chlorhexidine added tomixtures containing a sugar derivative and a source of amino groupsunderwent increased color formation, attributed to the Maillardreaction. It is also known that use of chlorhexidine results in anincreased dental pellicle. Nordbo proposed that chlorhexidine resultedin tooth staining in two ways: first, by increasing formation ofpellicle which contains more amino groups, and secondly, by catalysis ofthe Maillard reaction leading to colored products.

In accordance with this method, the compounds of Formula I areformulated into compositions adapted for use in the oral cavity.Particularly suitable formulations are oral rinses and toothpastesincorporating the active agent.

In the practice of this invention, conventional formulating techniquesare utilized with nontoxic, pharmaceutically acceptable carrierstypically utilized in the amounts and combinations that are well-knownfor the formulation of such oral rinses and toothpastes.

The agent of Formula I is formulated in compositions in an amounteffective to inhibit the formation of advanced glycosylationendproducts. This amount will, of course, vary with the particular agentbeing utilized and the particular dosage form, but typically is in therange of 0.01% to 1.0%, by weight, of the particular formulation.

Certain of the compounds encompassed by Formula I are novel compoundswhich can be prepared by modifications of chemical syntheses well-knownin the art. These compounds are those compounds of formula I wherein theR group is an amino group, with the proviso that when R' is a loweralkyl group, it is substituted as defined in formula I hereinabove andthe compounds of formula I wherein the R group is hydrogen, with theproviso that when R' is a lower alkyl group, it is substituted byhydroxy or a group of the formula ##STR9## wherein R" is hydroxy, alower alkoxy group, an aryl group optionally substituted by a loweralkoxy group, or a group of the formula --NR"'NH₂ wherein R"' ishydrogen or a methyl group. These compounds can be represented by thestructural formula (Ia): ##STR10## wherein when R is an amino group,then R' is a cycloalkyl group;

an aralkyl group wherein the alkyl portion contains from 1 to 6 carbonatoms, and the aryl portion is optionally substituted by one to threehydroxy, lower alkoxy or nitro groups;

a heteroaryl group containing from 1 to 3 heteroatoms selected from thegroup consisting of sulfur, oxygen and nitrogen;

a group of the formula ##STR11## wherein R" is hydroxy, a lower alkoxygroup, an aryl group optionally substituted by a lower alkoxy group, ora group of the formula --NR"'NH₂ wherein R"' is hydrogen or a methylgroup; or

a lower alkyl or alkylene group, optionally substituted by hydroxy or agroup of the formula ##STR12## wherein R" is hydroxy, a lower alkoxygroup, an aryl group optionally substituted by a lower alkoxy group, ora group of the formula --NR"'NH₂ wherein R"' is hydrogen or a methylgroup;

or when R is hydrogen; then

R' is a cycloalkyl group;

a group of the formula ##STR13## wherein R" is hydroxy, a lower alkoxygroup, an aryl group optionally substituted by a lower alkoxy group, ora group of the formula --NR"'NH₂ wherein R"' is hydrogen or a methylgroup; or

a lower alkyl group or an alkylene group, optionally substituted byhydroxy or a group of the formula ##STR14## wherein R" is hydroxy, alower alkoxy group, an aryl group optionally substituted by a loweralkoxy group, or a group of the formula --NR"'NH₂ wherein R"' ishydrogen or a methyl group;

with the proviso that when R' is a lower alkyl group, then it must besubstituted;

and their biologically or pharmaceutically acceptable acid additionsalts.

The following compounds of the present invention are thus novel andheretofore unknown in the prior art:

3,4-diamino-6-(3,4-dimethoxystyryl)-1,2,4-triazine-5(4H)-onehydrochloride;

3,4-diamino-6-(2'-carboxyethyl)-1,2,4-triazine-5(4H)-one hydrochloride;

3,4-diamino-6-cyclopropyl-1,2,4-triazine-5(4H)-one hydrochloride;

3,4-diamino-6-carboxyethyl-1,2,4-triazine-5(4H)-one hydrochloride;

3,4-diamino-6-(2'-ethoxycarbonylethyl)-1,2,4-triazine-5(4H)-onehydrochloride;

3,4-diamino-6-(1'-ethoxycarbonylethyl)-1,2,4-triazine-5(4H)-onehydrochloride;

3,4-diamino-6-(1'-ethoxycarbonylheptyl)-1,2,4-triazine-5(4H)-onehydrochloride;

3,4-diamino-6-(3'-methoxyphenylcarbonylmethylene)-1,2,4-triazine-5(4H)-one;

6-(3,4-diamino-5(4H)-one-1,2,4-triazinyl)hydrazide;

6-(3,4-diamino-5(4H)-one-1,2,4triazinyl)hydrazide hydrochloride;

3-[6'-(3',4'-diamino-5'(4H)-one-1',2',4'-triazinyl)]-propionichydrazide;

2-methyl-6'-(3',4'-diamino-5(4H)-one-1,2,4-triazinyl)hydrazide;

3,4-diamino-6-(1',1'-dimethyl-2'-hydroxyethyl)-1,2,4-triazine-5(4H)-onehydrochloride; and

4-amino-3-hydrazino-6-(4-hydroxybenzyl)-1,2,4-triazine-5(4H)-one.

The compounds of formula I can be prepared according to the methodsdescribed in Dornow et al., Chem. Ber. 97:2647 and 2173 (1964), Veda etal., Chem. Pharm. Bul. (1964), 12, 100, J. Chem Soc., Perkin Trans.,1(12), 2037-2049 (1986). or those of U.S. Pat. Nos. 4,036,632 and3,961,936, or as shown in the various schemes below. These references,in particular, describe the preparation of the compounds of formula Iwherein R is hydrogen.

The triazines of formula I wherein R' is other than a lower alkyl groupsubstituted by a C(O)R wherein R" is a hydrazine or methylhydrazinegroup can be prepared by the synthetic route shown below in Scheme I.

In the synthetic route shown as Scheme I, the appropriate pyruvic acidderivative of formula II wherein R'* is a cycloalkyl group; an aralkylgroup wherein the alkyl portion contains from 1 to 6 carbon atoms, andthe aryl portion is optionally substituted by 1 to three hydroxy, loweralkoxy or nitro groups; a heteroaryl group containing from 1 to 3heteroatoms selected from the group consisting of sulfur, oxygen andnitrogen; a group of the formula ##STR15## wherein R" is hydroxy, alower alkoxy group, an aryl group optionally substituted by a loweralkoxy group;

a lower alkyl group, optionally substituted by hydroxy or a group of theformula ##STR16## wherein R" is hydroxy, a lower alkoxy group, an arylgroup optionally substituted by a lower alkoxy group, and X is ahydroxy, ethoxy or sodium salt of the hydroxide, is reacted with a1,3-diaminoguanidine acid addition salt to afford the desired compoundof formula I wherein R' is other than a lower alkyl group substituted bya C(O)R" wherein R" is a hydrazine or methylhydrazine group. Thisreaction is typically conducted in an alkanol or aqueous alkanolsolvent, at the reflux temperature of the solvent system. Typicalreaction times vary according to the precise nature of the particularreactants, but are usually in the range of 12-36 hours. ##STR17##

The compounds of formula I wherein R' is a lower alkyl group substitutedby a C(O)R" wherein R" is a hydrazine or methylhydrazine group can beprepared by the synthetic route shown below in Scheme II.

In this synthetic route, a compound of formula I wherein R' is analkoxycarbonyl group is utilized as the starting material to prepare thecorresponding compound of formula I wherein R' is a lower alkyl groupsubstituted by a C(O)R" wherein R" is a hydrazine or methylhydrazinegroup. Typically, the compound of formula I wherein R' is analkoxycarbonyl group is treated with anhydrous hydrazine ormethylhydrazine and water at reflux temperatures. Typical times varyfrom about 0.5 to 3 hours. Preferably, the resultant product, which isthe free base, is converted to the acid addition salt, preferably onesuch as the hydrochloride, without isolation. The salt form is thencrystallized to give the desired product. ##STR18##

The compounds of formula I wherein R' is a lower alkyl group substitutedby a hydroxy group can also be prepared by the synthetic route shown inScheme III. In this scheme, a dihydro-4,4-dimethyl-2,3-furandione isutilized in place of the compound of formula II of Scheme I. Otherreaction conditions are generally similar. Thus afforded are theparticular hydroxyalkyl compounds wherein the lower alkyl group is anethyl or propyl group, optionally substituted by one or more lower alkylgroups. ##STR19##

The following examples are illustrative of the invention.

EXAMPLE 1 General Procedure for the Synthesis of 3,4-diamino-6-substituted-1,2,4-triazin-5(4H)-ones

A mixture of 1,3-diaminoguanidine monohydrochloride (10 mmole) and theappropriate pyruvic acid derivative of formula II (10 mmole) wererefluxed in ethanol (25 ml). Water was added dropwise until the reactionmixture formed a clear solution. This solution was then refluxedovernight, and then cooled to room temperature. The desired product,which separated upon standing, was filtered, dried and crystallized froman ethanol/water mixture. In the case where no product separated, themixture was evaporated to dryness and the residue was crystallized fromethanol/water mixture.

When the sodium salt of the pyruvic acid derivative of formula II wasused as a starting material, the resultant product was usually in theform of the free base. This free base form could then be converted intothe corresponding hydrochloride salt by treatment of the free base with2N hydrochloric acid.

The reaction was typically carried out using ethanol as the solventsystem when the derivative of pyruvic acid of formula II was an ester(X=ethoxy) was used.

Using the following pyruvic acid derivatives of formula II as startingmaterials, the compounds of formula I, described in Table 1, wereprepared:

(1) pyruvic Acid

(2) 4-hydroxyphenylpyruvic acid

(3) 2-ketobutyric acid

(4) phenylpyruvic acid

(5) 2-thiopheneglyoxylic acid

(6) 2-(3,4dimethoxystyryl)glyoxylic acid

(7) 2-nitrophenyl pyruvic acid

(8) 2-ketoglutaric acid

(9) 2-methyl-2-oxobutanoic acid, sodium salt

(10) 4methyl-2-oxobutanoic acid, sodium salt

(11) 2-oxopentanoic acid, sodium salt

(12) 2-oxohexanoic acid, sodium salt

(13) (±)-3-methyl-2-oxopentanoic acid, sodium salt

(14) cyclopropylglyoxylic acid

(15) ethyl 2-oxo-4-phenylbutyrate

(16) diethyl ketomalonate

(17) diethyl 2-oxoglutarate

(18) diethyl oxalpropionate

(19) diethyl 3-heptyl-2-oxosuccinate

(20) ethyl 3-(3-methoxybenzoyl) pyruvate

(21) glyoxylic acid

The physical data of the corresponding triazines (1-21) is given inTable I below. (X indicates the number of moles of the hydrochloridesalt in the product)

                                      TABLE 1                                     __________________________________________________________________________    Physical Data of Triazine Compounds                                           NO. R'                   R.sup.2                                                                         X M.P. °                                                                        Yield %                                   __________________________________________________________________________     1  H.sub.3 C--          H 0 260    90                                           -  2                                                                                                             H 1 278-279 (dec) 80                       -  3 H.sub.3 C--CH.sub.2 -- H 1 239-240 (dec) 48                              -  4                                                                                                             H 1 218-222 (dec) 33                       -  5                                                                                                             H 1 265-266 (dec) 21                       -  6                                                                                                             H 1 245-246 16                             -  7                                                                                                             H 1 239-241 (dec) 43                       -  8 HOOC--CH.sub.2 --CH.sub.2 -- H 0 247-248 (dec) 45                        9 (CH.sub.3).sub.2 --CH.sub.2 -- H 1 166-168 22                              10 (CH.sub.3).sub.2 --CH--CH.sub.2 -- H 0 190-192 48                          11 (CH.sub.3).sub.2 --CH--CH.sub.2 H 1 260-262 (dec) 59                       12 H.sub.3 C--CH.sub.2 --CH.sub.2 -- H 1 189-191 73                           13 H.sub.3 C--(CH.sub.2).sub.3 -- H 1 166-167 36                               - 14                                                                                                             H 0 189-191 65                             - 15                                                                                                             H 0 213-214 (dec) 17                       - 16                                                                                                             H 1 225-226 62                             - 17                                                                                                             H 1 227-229 (dec) 64                       - 18                                                                                                             H 1 205-207 53                             - 19                                                                                                             H 1 185-186 39                             - 20                                                                                                             H 1 185-186 31                             - 21                                                                                                             H 1 185-187 52                          __________________________________________________________________________

EXAMPLE 2 Synthesis of 6(3,'4'-diamino-5-one-1,2,4-triazinyl)-hydrazide

3,4-Diamino-6-carboxyethyl-1,2,4-triazin-5(4H)-one hydrochloride (1.59,6.36 mmole) was taken in methanol (15 ml). Anhydrous hydrazine (156.25mmole) and water (1 ml) were added and refluxed for 1 hour. On coolingto room temperature, the product which separated was filtered, dried andcrystallized from ethanol to give6-(3',4'-diamino-5(4H)-one-1,2,4-triazinyl)hydrazide, in a yield of 60%,to give a product having a m. p. of >340° C.

This compound was converted into its dihydrochloride by treatment with2N hydrochloric acid. The product,6-(3',4'-diamino-5(4H)-one-1,2,4-triazinyl)hydrazide dihydrochloride,was crystallized from ethanol/water mixture in 77% yield, and exhibits am. p. of 134-135 ° C. (dec).

Using same procedure with methyhydrazine as a starting material,2-methyl-[6'-(3',4'-diamino-5(4H)-one-1,2,4-triazinyl)]hydrazide, m. p.126-128°, and2-methyl-[6'-(3',4'-diamino-5(4H)-one-1,2,4-triazinyl)]hydrazidedihydrochloride, m. p. 178-180° (dec) were prepared.

EXAMPLE 3 Synthesis of3,4-diamino-6-(1',1'-dimethyl-2'-hydroxyethyl)-1,2,4-triazin-5(4H)-onehydrochloride

Dihydro-4,4-dimethyl-2,3-furandione (2 g, 15.6 mmole) and1,3-diaminoguanidine monohydrochloride (12.96 g, 15.61 immole) weredissolved in ethanol and the resulting solution heated to refluxtemperatures. To the refluxing reaction mixture, was added water until aclear solution was obtained. The mixture was then refluxed overnight,cooled to room temperature and stored at -20° C. overnight. The productwhich separated was filtered and dried. It was crystallized from amixture of ethanol and water to give3,4-diamino-6-(1',1'-dimethyl-2'-hydroxyethyl)-1,2,4,-triazin-5(4H)-onehydrochloride (2.24 g, 61%), m.p. 221-222° C. (dec).

EXAMPLE 4 Synthesis of4-amino-3-hydrazino-6-(4'-hydroxybenzyl)-1,2,4-triazin-5(4H)-onehydrochloride

4-Hydroxyphenylpyruvic acid (2 g, 11 mmole) and triaminoguanidinemonohydrochloride (2.21 g, 15.78 mmole) were taken up in ethanol (25 ml)and heated until reflux. A few drops of water was added until thereaction mixture was clear. The resultant mixture was refluxed 24 hoursand cooled to room temperature. The solid which separated was filtered,dried and crystallized from ethanol/water to give the title product (2g, 47%), m.p. 209-210° C. (dec).

In a similar manner,4-amino-3-hydrazino-6-methyl-1,2,4-triazin-5(4H)-one hydrochloride, m.p.195-196 ° C. and 4-amino-3-hydrazino-1,2,4-triazin-5(4H)-onehydrochloride, m.p. 260-261° C., were prepared.

EXAMPLE 5

The following method was used to evaluate the ability of the compoundsof the present invention to inhibit the cross-linking of glycated bovineserum albumin (AGE-BSA) to the rat tail tendon collagen coated 9-wellplate.

The AGE-BSA was prepared by incubating BSA at a concentration of 200 mgper ml with 200 mM glucose in 0.4 M sodium phosphate buffer, pH 7.4 at37° C. for 12 weeks. The glycated BSA was then extensively dialyzedagainst phosphate buffer solution (PBS) for 48 hours with additional 5times buffer exchanges. The rat tail tendon collagen coated plate wasblocked first with 300 μl of superbloc blocking buffer (Pierce #37515X)for one hour. The blocking solution was removed from the wells bywashing the plate twice with PBS-Tween 20 solution (0.05% Tween 20)using a NUNC-multiprobe or Dynatech ELISA-plate washer. Cross-linking ofAGE-BSA (1 to 10 μg per well depending on the batch of AGE-BSA) to rattail tendon collagen coated plate was performed with and without thetesting compound dissolved in PBS buffer at pH 7.4 at the desiredconcentrations by the addition of 50 μl each of the AGE-BSA diluted inPBS or in the testing compound at 37° C. for 4 hours. The unbrowned BSAin PBS buffer with or without testing compound were added to theseparate wells as the blanks. The un-cross-linked AGE-BSA was thenremoved by washing the wells three times with PBS-Tween buffer. Thecross-linked AGE-BSA to the tail tendon coated plate was thenquantitated by the polyclonal antibody raised against AGE-RNase. After aone-hour incubation period, AGE antibody was removed by washing 4 timeswith PBS-Tween.

The bound AGE antibody was then detected with the addition ofhorseradish peroxidase-conjugated secondary antibody, e.g., goatanti-rabbit immunoglobulin and incubation for 30 minutes. The substrateof 0.1M citrate buffer containing 0.03% H₂ O₂ (2 yield #00-2008) and2,2-azino-di(3-ethylbenzthiazoline sulfonic acid) (ABTS chromogen)(Zymed #00-2011) was added. The reaction was allowed for an additional15 minutes and the absorbance was read at 410 nm in a Dynatech platereader.

The % inhibition of each test compound was calculated as follows.

    % inhibition={[Optical density (without compound)-optical density (with compound)]/optical density (without compound)]}100%

The IC₅₀ in mM are as follows:

    ______________________________________                                        Test Compound           IC.sub.50                                             ______________________________________                                        3,4-diamino-6-methyl-1,2,4-                                                                           0.13                                                    triazine-5(4H)-one;                                                           3,4-diamino-6-(4-hydroxybenzyl)- 1.7                                          1,2,4-triazine-5(4H)-one hydrochloride                                        3,4-diamino-6-ethyl-1,2,4- 1.54                                               triazine-5(4H)-one hydrochloride                                              3,4-diamino-6-benzyl-1,2,4- 11.4                                              triazine-5(4H)-one hydrochloride                                              3,4-diamino-6-(2'-carboxyethyl)- 1.6                                          1,2,4-triazine-5(4H)-one                                                      3,4-diamino-6-(2-methypropyl)- 5.1                                            1,2,4-triazine-5(4H)-one                                                      3,4-diamino-6-(1'-ethoxycarbonylethyl-) 1.85                                  1,2,4-triazine-5(4H)-one hydrochloride                                        3,4-diamino-6-(1'-ethoxycarbonylheptyl-) 6.6                                  1,2,4-triazine-5(4H)-one hydrochloride                                        6-(3',4'-diamino-5(4H)-one-1,2,4- 0.73                                        triazinyl)hydrazide                                                           3-[6'-(3',4'-diamino-5'(4H)-one- 0.5                                          1',2',4'-triazinyl)]-propionic hydrazide                                      hydrochloride                                                                 2-methyl-6'-(3',4'-diamino-5(4H)-one- 3.5                                     1,2,4-triazinyl)hydrazide                                                     4-amino-3-hydrazino-6-methyl-1,2,4- 0.016                                     triazine-5(4H)-one hydrochloride                                              4-amino-3-hydrazino-6-(4-hydroxybenzyl)- 0.45                                 1,2,4-triazine-5(4H)-one hydrochloride                                        4-amino-3-hydrazino-1,2,4-triazine- 1.56                                      5(4H)-one hydrochloride                                                     ______________________________________                                    

The above experiment suggests that this type of drug therapy hasbenefits in reducing the pathology associated with the advancedglycosylation of proteins and the formation of cross-links betweenproteins and other macromolecules. Drug therapy may be used to preventthe increased trapping and cross-linking of proteins that occurs indiabetes and aging which leads to sequelae such as retinal damage, andextra-vascularly, damage to tendons, ligaments and other joints.

EXAMPLE 6

The following method was used to evaluate the ability of the compoundsof the present invention to inhibit the cross-linking of N-acetylglycyl-lysinc methyl ester in the presence of ribose.

Materials:

N-acetylglycyllysine methyl ester (DP in formula below)

Ribose (R in formula below)

Test compounds (C in formula below)

Reagents:

0.5M sodium phosphate buffer pH 7.4

N-acetylglycyllysine methyl ester in 0.5M sodium phosphate buffer, pH7.4

Ribose: 800 mM

Test compounds dissolved in the above buffer and the pH is adjusted to7.4, if necessary

Procedure:

Reaction mixtures are prepared as follows:

    ______________________________________                                        80 mg/ml N-acetylglycyllysine                                                                   0.1       0.1   --                                            methyl ester/buffer                                                           ribose 0.1 0.1 0.1                                                            test compound -- 0.1 0.1                                                      buffer 0.2 0.1 0.2                                                          ______________________________________                                    

and incubated at 37° C. for 16-24 hours. At the end of the incubationperiod reaction mixture is diluted with 3 ml distilled water and thefluorescence is read using an excitation wavelength of 350 nm andemission wavelength of 400 nm. The inhibition of the cross-linking iscalculated from the decrease in the fluorescence in the presence of thetest compounds according to the formula:

    Inhibition (%)=100×[DPRC fluorescence-RC fluorescence]/DPR fluorescence

The Inhibition by various test compounds (IC₅₀) is as follows:

    ______________________________________                                        Test compound          IC.sub.50 mM                                           ______________________________________                                        3,4-diamino-6-methyl-1,2,4-                                                                          1.23                                                     triazine-5(4H)-one;                                                           3,4-diamino-6-(4-hydroxybenzyl)- 4.4                                          1,2,4-triazine-5(4H)-one hydrochloride                                        3,4-diamino-6-ethyl-1,2,4- 2                                                  triazine-5(4H)-one hydrochloride                                              3,4-diamino-6-benzyl-1,2,4- 1.2                                               triazine-5(4H)-one hydrochloride                                              3,4-diamino-6-(3,4-dimethoxystyryl)- 0.35                                     1,2,4-triazine-5(4H)-one hydrochloride                                        3,4-diamino-6-(2'-nitrobenzyl)- 0.9                                           1,2,4-triazine-5(4H)-one hydrochloride                                        3,4-diamino-6-(2'-carboxyethyl)- 1.97                                         1,2,4-triazine-5(4H)-one                                                      3,4-diamino-6-isopropyl-1,2,4- 1.05                                           triazine-5(4H)-one hydrochloride                                              3,4-diamino-6-(2-methypropyl)- 1.16                                           1,2,4-triazine-5(4H)-one                                                      3,4-diamino-6-propyl-1,2,4- 1.3                                               triazine-5(4H)-one hydrochloride                                              3,4-diamino-6-butyl-1,2,4- 1.6                                                triazine-5(4H)-one                                                            3,4-diamino-6-(1-methylpropyl)- 1.4                                           1,2,4-triazine-5(4H)-one                                                      3,4-diamino-6-cyclopropyl-1,2,4- 2                                            triazine-5(4H)-one                                                            3,4-diamino-6-phenethyl-1,2,4- 2.7                                            triazine-5(4H)-one hydrochloride                                              3,4-diamino-6-carboxyethyl-1,2,4- 0.72                                        triazine-5(4H)-one hydrochloride                                              3,4-diamino-6-(2'-ethoxycarbonylethyl- 0.84                                   1,2,4-triazine-5(4H)-one hydrochloride                                        3,4-diamino-6-(1'-ethoxycarbonylethyl- 1.65                                   1,2,4-triazine-5(4H)-one hydrochloride                                        3,4-diamino-6-(1'-ethoxylcarbonylheptyl- 0.48                                 1,2,4-triazine-5(4H)-one hydrochloride                                        3,4-diamino-6-(3'-methoxyphenylcarbonyl- >10                                  methylene)-1,2,4-triazine-5(4H)-one                                           hydrochloride                                                                 6-(3',4'-diamino-5(4H)-one-1,2,4- 0.73                                        triazinyl)hydrazide                                                           3-[6'-(3',4'-diamino-5'(4H)-one- 1.5                                          1',2',4'-triazinyl)]-propionic hydrazide                                      hydrochloride                                                                 2-methyl-6'-(3',4'-diamino-5(4H)-one- 0.83                                    1,2,4-triazinyl)hydrazide                                                     3,4-diamino-6-(1',1'-dimethyl-2'- 0.97                                        hydroxyethyl)-1,2,4-triazine-5(4H)-one                                        4-amino-3-hydrazino-6-methyl-1,2,4- <3                                        triazine-5(4H)-one hydrochloride                                              4-amino-3-hydrazino-6-(4-hydroxybenzyl)- >1                                   1,2,4-triazine-5(4H)-one hydrochloride                                        4-amino-3-hydrazino-1,2,4-triazine- >10                                       5(4H)-one hydrochloride                                                     ______________________________________                                    

The above experiment suggests that this type of drug therapy will reducethe pathology associated with the advanced glycosylation of proteins andthe formation of cross-links between proteins and other macromolecules.Drug therapy may be used to prevent the increased trapping andcross-linking of proteins that occurs in diabetes and aging which leadsto sequelae such as retinal damage, and extra-vascularly, damage totendons, ligaments and other joints.

EXAMPLE 7

    ______________________________________                                        Tablet            mg/tablet                                                   ______________________________________                                        Compound of Formula I                                                                           50                                                            Starch 50                                                                     Mannitol 75                                                                   Magnesium stearate 2                                                          Stearic acid 5                                                              ______________________________________                                    

The compound, a portion of the starch and the lactose are combined andwet granulated with starch paste. The wet granulation is placed on traysand allowed to dry overnight at a temperature of 45° C. The driedgranulation is comminuted in a comminutor to a particle size ofapproximately 20 mesh. Magnesium stearate, stearic acid and the balanceof the starch are added and the entire mix blended prior to compressionon a suitable tablet press. The tablets are compressed at a weight of232 mg. using a 11/32" punch with a hardness of 4 kg. These tablets willdisintegrate within a half hour according to the method described in USPXVI.

EXAMPLE 8

    ______________________________________                                        Lotion           mg/g                                                         ______________________________________                                        Compound of Formula I                                                                           1.0                                                           Ethyl alcohol 400.0                                                           Polyethylene glycol 400 300.0                                                 Hydroxypropyl cellulose  5.0                                                  Propylene glycol to make 1.0 g                                              ______________________________________                                    

EXAMPLE 9

    ______________________________________                                        Oral Rinse                                                                    ______________________________________                                        Compound of Formula I: 1.4%                                                     Chlorhexidine gluconate 0.12%                                                 Ethanol 11.6%                                                                 Sodium saccharin 0.15%                                                        FD&C Blue No. 1 0.001%                                                        Peppermint Oil 0.5%                                                           Glycerine 10.0%                                                               Tween 60 0.3%                                                                 Water to 100%                                                               ______________________________________                                    

EXAMPLE 10

    ______________________________________                                        Toothpaste                                                                    ______________________________________                                        Compound of Formula I:   5.5%                                                   Sorbitol, 70% in water 25%                                                    Sodium saccharin  0.15%                                                       Sodium lauryl sulfate  1.75%                                                  Carbopol 934, 6% dispersion in 15%                                            Oil of Spearmint  1.0%                                                        Sodium hydroxide, 50% in water  0.76%                                         Dibasic calcium phosphate dihydrate 45%                                       Water to 00%                                                                ______________________________________                                    

EXAMPLE 11

To further study the ability of inhibitors of nonenzymatic browning toprevent the discoloration of protein on a surface, such as that whichoccurs on the tooth surface, the following surface browning experimentis performed. As a substitute for a pellicle-covered tooth surface,unexposed and developed photographic paper is used to provide a fixedprotein (gelatin, i.e., collagen) surface on a paper backing. Fivemillimeter circles are punched and immersed for one week at 50° C. in asolution of 100 mM glucose-6-phosphate in a 0.5M phosphate buffer, pH7.4 containing 3 mM sodium azide. Glucose-6-phosphate is a sugar capableof participating in nonenzymatic browning at a more rapid rate thanglucose. In addition to the glucose-6-phosphate, chlorhexidine and/or acompound of Formula I are included. After incubation, the gelatin/paperdisks are rinsed with water, observed for brown color, and photographed.Incubation of the disks in glucose-6-phosphate alone shows slight browncolor versus disks soaked in buffer alone. Inclusion of chlorhexidine(in the form of Peridex® at a final concentration of 0.04%chlorhexidine) shows significant browning. Addition of a compound ofFormula I to the chlorhexidine completely inhibits browning of thegelatin, as does inclusion of a compound of Formula I in the absence ofchlorhexidine.

The slight brown color formed by the action of glucose-6-phosphate onthe gelatin surface alone and its prevention by a compound of Formula Idemonstrates the utility of the present invention in preventingnonenzymatic browning of tooth surfaces. The enhanced browning in thepresence of chlorhexidine and its prevention with a compound of FormulaI demonstrates the utility of the present invention in preventing theanti-plaque agent-enhanced nonenzymatic browning which occurs withchlorhexidine.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

What is claimed is:
 1. The compound which is3,4-diamino-6-(4-hydroxybenzyl)-1,2,4-triazine-5(4H)-one hydrochlorideor another pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition for administration to an animal to inhibit the advancedglycosylation of a target protein within said animal, comprising apharmaceutically effective amount of3,4-diamino-6-(4-hydroxybenzyl)-1,2,4-triazine-5(4H)-one hydrochlorideor another pharmaceutically acceptable salt thereof.